When a subendothelium is exposed due to an injury of vessel walls in a living body, platelets flowing through the bloodstream immediately adhere to the subendothelim. This triggers a series of platelet activation processes including platelet aggregation and release of intracellular granules, after which thrombus is formed, and thus bleeding is arrested. Accordingly, thrombus formation is necessary and indispensable for the physiological hemostatic mechanism. However, on the other hand, the thrombus causes thrombotic diseases such as myocardial infarction, angina pectoris, cerebral infarction, and cerebral thrombosis which become to hold higher ranks of the cause of death in proportion to the aging of society. Such a situation is recognized as a serious problem.
Many antithrombotic agents have been hitherto developed in order to cure and prevent the thrombotic diseases. However, problems to be solved remain in that many of the conventional antithrombotic agents still have low curative effectiveness in clinical application, they have low specificity to thrombus, and they cause hemorrhagic tendency as a side effect. One of the causes of such circumstances is considered as follows. Namely, almost all of the antithrombotic agents are designed only for the purpose of inhibiting the platelet-activating process. A method for measuring platelet aggregation in vitro, which provides an index of the activity, is insufficient to reflect the complicated thrombus formation process in vivo.
Thrombus formation proceeds in accordance with specific binding between glycoprotein on platelet membrane and subendothelium or proteins in plasma. Especially, glycoprotein IIb/IIIa (hereinafter abbreviated as "GPIIb/IIIa") on platelet membrane functions as a receptor for fibrinogen in the final stage of the thrombus formation. Accordingly, it is expected that GPIIb/IIIa-antagonists may be used as a potent antithrombotic agent. The fibrinogen-binding site on GPIIb/IIIa includes an RGD primary sequence of amino acids. As a result of synthesis and evaluation of many RGD derivatives, it has been confirmed that GPIIb/IIIa antagonist exhibits the antithrombotic effect by strongly inhibiting the platelet aggregation, according to an animal models in vivo and clinical investigations (Thrombosis and Haemostasis, vol. 69, p. 560, 1993). However, a problem emerges in that GPIIb/IIIa antagonists simultaneously suppress the normal hemostatic mechanism, and hence the hemorrhagic tendency as a side effect appears more strongly as compared with the conventional antithrombotic agents (The Lancet, vol. 343, p. 881, 1994; The New England Journal of Medicine, vol. 330, p. 956, 1994).
On the other hand, those known as important proteins which function at the early stage of thrombus formation include glycoprotein Ib on platelet membrane (hereinafter abbreviated as "GPIb") and von Willebrand factor in blood plasma (hereinafter abbreviated as "vWF"). Hemorrhagic lesions associated with occurrence of qualitative and quantitative change in vWF include von Willebrand disease (hereinafter referred to as "vWD"). A clinical knowledge has been obtained that serious bleeding scarcely occurs in vWD patients as compared with patients of thrombasthenia (hemorrhagic disease due to deficiency of GPIIb/IIIa). Therefore, a possibility is conceived that powerful antithrombotic action may be exhibited without involving the hemorrhagic tendency by inhibiting the interaction between GPIb and vWF. However, only a monoclonal antibody and a low molecular weight compound ATA (Aurin Tricarboxylic Acid; Blood, vol. 72, p. 1898, 1988) have been known as substances to specifically inhibit the interaction between GPIb and vWF. Any antithrombotic action of the anti-GPIb monoclonal antibody in vivo has not been confirmed. Instead, side effects are emphasized in that the anti-GPIb monoclonal antibody causes thrombocytopenia, and it prolongs the bleeding time (Blood, vol. 70, 344a, 1987; Jpn. J. Clin. Pathol., vol. 40, p. 266, 1992). Further, it has been reported for those which antagonize vWF that ATA described above and a mouse anti-swine vWF monoclonal antibody BB3-BD5 exhibit antithrombotic efficacies in an in vivo experiment with animals (Circulation, vol. 81, p. 1106, 1990). However, side effects cannot be neglected in the case of both ATA and BB3-BD5. Namely, ATA exhibits the antithrombotic action by inhibiting the interaction between GPIb and vWF, while ATA simultaneously involves completely opposite side effects such that it enhances platelet aggregation and release reaction caused by the aid of collagen, arachidonic acid, A23187, PAF, and TXA.sub.2 (Thrombosis and Haemostasis, vol. 68, p. 189, 1992). On the other hand, BB3-BD5 exhibits a strong hemorrhagic tendency in its antithrombotic dose (Proc. Natl. Acad. Sci. USA, vol. 84, p. 8100, 1987; SURGERY, vol. 112, p. 433, 1992).
As described above, there is a dilemma in the existing antithrombotic agents in that the antithrombotic action as an medicinal effect cannot be separated from the hemorrhagic tendency as a side effect (there is no difference between the medicinally effective amount and the amount to cause the side effect).
Recently, shear stress-induced platelet aggregation (hereinafter abbreviated as "SIPA") attracts attention, as closely related to thrombus formation in a pathological state. The vascular diameter is small, and the bloodstream has a large velocity in arteriosclerosis lesions and small arteries. Therefore, a high shear stress occurs in such regions due to the interaction between vessel wall and blood. In such a situation, vWF in blood is activated, and its tertiary structure is changed. As a result, vWF plays a crucial role in thrombus formation. Namely, the following process is known. Firstly, vWF existing on subendothelium binds to GPIb on platelet membrane, and thus platelets adhere to vessel wall. Secondly, vWF existing in blood plasma cross-links glycoprotein IIb/IIIa on platelet membrane, and thus the platelet aggregation reaction is allowed to proceed. Consequently, thrombus formation finally occurs.
It is generally known that an antibiotic ristocetin or a snake venom botrocetin allows vWF to cause a change in tertiary structure in vitro, equivalent to the change under a high shear stress. Namely, in the presence of ristocetin or botrocetin, vWF acquires the binding ability to GPIb. Methods for measuring the physiological activity of vWF in vitro by utilizing the foregoing characteristic include ristocetin-induced platelet aggregation (hereinafter abbreviated as "RIPA") and botrocetin-induced platelet aggregation (hereinafter referred to as "BIPA"), as well as a method for measuring binding of vWF to GPIb in the presence of ristocetin or botrocetin. The foregoing methods are widely utilized. Owing to the progress of experimental techniques, an apparatus has been also developed, in which SIPA is measured in vitro by actually applying a shear stress. It is considered that an identical domain on vWF involved in the binding to GPIb in any of the reactions.
Several antibodies against vWF, which inhibit the activity of vWF in vitro, have been hitherto obtained. However, many of them are inferior in reaction specificity, and almost all of them do not inhibit the botrocetin-dependent reaction, even though they inhibit the ristocetin-dependent reaction. As described above, it is considered that the GPIb-binding site on vWF induced by ristocetin is homologous to that induced by botrocetin. Therefore, the foregoing antibodies possibly recognize the binding site on vWF for ristocetin or botrocetin. Strictly speaking, it is possible to say that they do not inhibit the physiological activity of vWF, and hence they have low reaction specificities. In such circumstances, it has been reported that two antibodies, i.e., NMC-4 produced by Fujimura et al. (J. Nara Med. Assoc., vol. 36, p. 662, 1985) and RFF-VIIIRAG:1 produced by Tuddenham et al., inhibit in vitro the reaction depending on both of ristocetin and botrocetin (Blood, vol. 17, No. 1, p. 113, 1991).
It has been reported that epitopes for the two antibodies exist in the GPIb-binding site of the vWF molecule, and they are located between 449th and 728th amino acid residues of an amino acid sequence of the vWF molecule. Further, binding of iodine-labeled NMC-4 to vWF is partially inhibited by RFF-VIIIRAG:1. According to this fact, it is considered that the both epitopes are located at positions considerably close to one another. Moreover, RFF-VIIRAG:1 inhibits BIPA only partially, while NMC-4 completely inhibits BIPA. For this reason, studies have been diligently made in the scientific field of vWF by using NMC-4, and certain results have been obtained. Among animals other than human, NMC-4 has its reactivity only with rat vWF.
When a monoclonal antibody against human vWF is prepared in order to obtain information on the GPIb-binding site of human vWF, or in order to use the monoclonal antibody as a preventive agent and a therapeutic agent against diseases relevant to vWF, it is considered to be desirable to prepare the monoclonal antibody as one having high specificity to human vWF.
On the other hand, when a new medicine is developed in an ordinary manner, it is unallowable to perform any test with human without previously performing a test with animals. When a test is performed in relation to physiological activities of vWF and anti-vWF monoclonal antibodies in vivo, it is necessary to use a monoclonal antibody which makes it possible to perform a test with animals, i.e., a monoclonal antibody simultaneously having reactivity with vWF of an animal other than human. By the way, GPIIb/IIIa antagonists, which strongly suppresses human platelet aggregation by the aid of fibrinogen, are not effective on rat (Thrombosis and Haemostasis, vol. 70, p. 531, 1993). Further; rat does not cause ristocetin-induced aggregation. According to these facts, it is generally considered that the mechanism of thrombus formation greatly differs between rat and human. Therefore, it is almost meaningless to evaluate the antithrombotic action of any anti-vWF antibody by using rat. On the contrary, in the case of guinea pig, platelet aggregation is suppressed by GPIIb/IIIa antagonists. Further, ristocetin-induced aggregation is also induced in the same manner as human. Accordingly, it is considered that guinea pig is most suitable as an animal thrombus model for in vivo experiments when the antithrombotic action is evaluated.
According to the foregoing viewpoints, any of a monoclonal antibody having reactivity with only human vWF, and a monoclonal antibody having reactivity with both human vWF and guinea pig vWF is useful. However, such anti-human vWF monoclonal antibodies are not known.
Further, an anti-human vWF monoclonal antibody, which has been confirmed to have antithrombotic action in vivo, is not known.